The RNA Interference Of ACC Synthase Gene In Eustoma Grandiflorum 'Double Mariachi Pink'

Eustoma grandiflorum is Gentianceae biennial herbs, which was one of the popular cut flowers in the world, and has high economic, viewing and admiring value.Flower's viewing life, especially the vase life of cut flower, is an extremely important quality, which directly affect the commodity value of the flowers. How to extend the viewing life of flowers has been the key issue concerned the experts at home and abroad.Ethylene is a senescence hormone, ACC synthase (ACS) is its biosynthesis rate-limiting enzyme. In order to delay the senescence of the cut flowers, one of the methods is to inhibit the biosynthesis of ethylene. In the study, we use Eustoma grandiflorum 'Double Mariachi Pink' variety as material. Through the ACS interference vector construction and transformation to the plants, we expect to cultivate a new variety of Eustoma grandiflorum which has longer vase life.First, useing Eustoma grandiflorum genomic DNA as templat, we get ACS activity sequence through PCR. The sequence length is a part of full-length genes, with the length of 1137bp, including the reaction active center of ACS which related to the combination and catalysis with the pyridoxal phosphate coenzyme. In order to construct the interference vector pCAMBIA1301-gfp-(GUS+ACS) i-NPTII, put the sequence into the vector pCAMBIA1301-gfp-(GUS+X) i-NPTII which contains the bidirectional promoter after digestion. The interference vector carries NPTII as a marker gene, gfp as a reporter gene.Secondly, we infect Eustoma grandiflorum through the Agrobacterium-mediated transformation system with the interference vector.The infection conditions as follows: Resuspend the Eustoma grandiflorum seedlings' leaves which have been cultured for three days with the concentration of OD600≈0.5 of Agrobacterium for 10min, then culture five days in the dark under the conditions of 24℃, and then transferred to selection medium for selected training.Finally, use 112 resistant seedlings for root testing.64 transformed seedlings rooted out of 112 resistant seedlings. The rooting rate is 57.1%. Design a pair of primers based on the GFP reporter gene sequence for the PCR analysis. The test demonstrates that 18 transformed plants are positive. In addition, based on some existing studies about the relevant factors which affecting the transplantation survival rate of Eustoma grandiflorum, such as culture media, openings hardening time, the effective number of leaves and the handling of the root washing, etc., we do some studies from the following two aspects:the selection of transplanation time impacting on the survival rate and the height of the seedlings impacting on bloom. The studies lay the foundation for the further identification of transgenic plants.